|
| Status |
Public on Feb 10, 2009 |
| Title |
Macrophage_dendriplex_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Macrophages, control2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: macrophages
treatment: 5 hours of incubation
|
| Treatment protocol |
Incubated for 5 hours in RPMI medium with the same enrichment.
|
| Growth protocol |
PBMCs obtained from healthy individuals, were processed by Ficoll density gradient centrifugation and activated with 60 u/mL of IL2 and 2 µg/mL of phytohemaglutinine for 72h in RPMI medium enriched with 10% of fetal bovine serum and 1 % of ampicillin, 1% of cloxacillin, 0.32% of gentamicin and 2nM glutamine at 37ºC in a 5% CO2 atmosphere. Macrophages were isolated from these cells mechanically
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA of cells was extracted using RNeasy Mini Kit (Qiagen) following manufacture’s instructions. RNA concentrations were measured using the Nanodrop Spectrophotometer ND-100 UV/Vis (Nanodrop Technologies).
|
| Label |
Alexa Fluor 555
|
| Label protocol |
For each sample, 1 µg of total RNA was amplified and labelled using SuperScript Indirect RNA Amplification Kit (Invitrogen) following manufacturer's instructions. Briefly, RNA was denatured and reverse transcribed to cDNA. This cDNA was purified and in vitro transcribed to RNA using T7 RNA polymerase. Amplified RNA was purified and quantified using a Nanodrop Spectrophotometer ND-100 UV/Vis (Nanodrop Technologies). Control and dendrimer probes were labelled, respectively, with Alexa Fluor 555 reactive dye decapack and Alexa Fluor 647 reactive dye decapack. Labeled RNA was again purified and quantified using a Nanodrop Spectrophotometer ND-100 UV/Vis (Nanodrop Technologies) and RNA quality checked using the Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| |
|
| Channel 2 |
| Source name |
Macrophages, dendriplex2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: macrophages
treatment: 5 hours exposure to dendriplex
|
| Treatment protocol |
Incubated for 5 hours in RPMI medium with the same enrichment and exposed to 2G-NN16 complexed with a random siRNA (dendriplex). For dendriplex formation, a ratio of charges 2:1 (NN16:siRNA) was used.
|
| Growth protocol |
PBMCs obtained from healthy individuals, were processed by Ficoll density gradient centrifugation and activated with 60 u/mL of IL2 and 2 µg/mL of phytohemaglutinine for 72h in RPMI medium enriched with 10% of fetal bovine serum and 1 % of ampicillin, 1% of cloxacillin, 0.32% of gentamicin and 2nM glutamine at 37ºC in a 5% CO2 atmosphere. Macrophages were isolated from these cells mechanically
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA of cells was extracted using RNeasy Mini Kit (Qiagen) following manufacture’s instructions. RNA concentrations were measured using the Nanodrop Spectrophotometer ND-100 UV/Vis (Nanodrop Technologies).
|
| Label |
Alexa Fluor 647
|
| Label protocol |
For each sample, 1 µg of total RNA was amplified and labelled using SuperScript Indirect RNA Amplification Kit (Invitrogen) following manufacturer's instructions. Briefly, RNA was denatured and reverse transcribed to cDNA. This cDNA was purified and in vitro transcribed to RNA using T7 RNA polymerase. Amplified RNA was purified and quantified using a Nanodrop Spectrophotometer ND-100 UV/Vis (Nanodrop Technologies). Control and dendrimer probes were labelled, respectively, with Alexa Fluor 555 reactive dye decapack and Alexa Fluor 647 reactive dye decapack. Labeled RNA was again purified and quantified using a Nanodrop Spectrophotometer ND-100 UV/Vis (Nanodrop Technologies) and RNA quality checked using the Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| |
|
| |
| Hybridization protocol |
Before the hybridization, RNA (1.65 µg) was fragmented using the Gene Expression Hybridization Kit (Agilent Technologies) by incubation with 25x Fragmentation Buffer and 10x Blocking Agent for 30 minutes at 60ºC. Fragmented amplified RNA was hybridized to Agilent 4x44K whole genome human chip (Agilent Technologies) in Hybridization Buffer of Gene Expression Hybridization kit using gasket slides (Hybridization Gasket Slide Kit - 4 microarrays per slide format, Agilent Technologies, CA), Agilent microarray hybridization chambers (G2534A) (Agilent Technology) and a DNA Microarray Hybridization Oven (Agilent Technology) for 17 hours at 65ºC and 10 r.pm. After this time, 2 washes of 1 minute each, the first with Wash Solution 1 and the second with Wash Solution 2 (Agilent Technology) were performed. The slides were centrifugated at 1000 r.p.m. for 2 min, before the scanning process.
|
| Scan protocol |
Slides were scanned at 10 μm resolution on a GenePix 4000B scanner (Axon Instruments, Inc., Foster City, CA) with independent excitation of the fluorophores Alexa Fluor 555 and Alexa Fluor 647. The resulting TIFF images were analyzed using GenePix Pro 4.0 software (Axon Instruments, Inc., Foster City, CA). Spots or areas of the array with obvious defects were manually flagged. The signal and background fluorescence intensities were calculated for each spot, and a GPR file for each hybridization was obtained
|
| Description |
Comparison between Control Macrophages and Macrophages exposed to dendriplex (2G-NN16 dendrimer complexed to a random siRNA).
|
| Data processing |
The quantification file was loaded into the Almazen database software (http://almazen.bioalma.cm). Background was subtracted and each experiment normalized by total intensity and sub-grid lowess.
|
| |
|
| Submission date |
Aug 11, 2008 |
| Last update date |
Oct 04, 2011 |
| Contact name |
Luis A. Lopez-Fernandez |
| E-mail(s) |
llopezf.hgugm@salud.madrid.org
|
| Phone |
34 914265026
|
| Organization name |
Hospital General Universitario Gregorio Marañon
|
| Department |
Pharmacy
|
| Lab |
Pharmacogenetics and Pharmacogenomics
|
| Street address |
Dr. Esquerdo 46
|
| City |
Madrid |
| ZIP/Postal code |
28007 |
| Country |
Spain |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE12405 |
Gene expression of human primary macrophages induced by carbosilane dendrimer 2G-NN16 complexed with or without siRNA |
|
